The primary goal of Project 2 (Caroline N. Dealy) will be to study the mechanisms involved in limb initiation. Previous studies indicate that IGF-1 is a key component of a medial to lateral signaling cascade that results in the establishment of the presumptive limb territories of the lateral plate and in the initial formation of the limb bud from these regions. FGF-10 has also recently been implicated in these processes. Studies on the role of IGF-1 in limb initiation will be expanded: the possible role of IGF binding proteins in the process will be explored; and the possible regulatory relationship between IGF-I and FGF-10 initial limb formation will be studied. TGF-alpha, like IGF-I and FGF-10 is specifically expressed in the mesoderm of the presumptive limb territories, but in the intervening non-limb forming flank. Thus, the possible role of TGF-alpha in limb initiation will be studied. TGF-alpha is also highly and specifically expressed in the presumptive AER, and so the possibility that it may play a role in determination or specification of the AER will be investigated. The homeobox-containing gene Dlx-5 is vertebrate homolog of the Drosophila Distal-less, which is one of the first genetic signals for limb formation in the fly embryo. Dlx-5 is highly and specifically expressed in the presumptive limb forming regions of the lateral plate of the chick embryo, but not in the flank. Furthermore, Dlx-5 expression is rapidly induced in the flank during the formation of FGF-induced ectopic limbs. Thus, the role of Dlx-5 in limb initiation will be studied, as will its possible regulatory relationships to IGF-I, FGF-10, and TGF-alpha in the process. A secondary goal of Projects will be to use the limb buds of the amelic mutant chick embryos limbless and wingless to explore the roles of FGFR2 signaling. Wnt3a, and Cux in regulating formation of the AER.